This application is to continue support for productive collaborative research done largely by the foreign PI, Laszlo Csanady, at Semmelweis University in Budapest, Hungary, to extend NIH grant R01 DK051767 of the US collaborator, David C. Gadsby. The parent grant aims to understand molecular mechanisms that control opening and closing CFTR (cystic fibrosis transmembrane conductance regulator) Cl- ion channels, encoded by the gene mutated in CF patients. CFTR is one of -50 human ABC (ATP-binding cassette) proteins, many (like SUR, the surfonylurea receptor, or P-glycoprotein and MRP, the multidrug resistance related protein) involved in human disease. CFTR is the sole ABC protein to form an ion channel, and so is the only one amenable to high-resolution studies of single-molecule function. But, statistical comparison of >15,000 available ABC sequences argues that certain functional mechanisms elucidated in CFTR will have broad applicability in other ABC proteins: this includes how ATP-driven events in the nucleotide-binding domains (NBDs) control conformational changes in the transmembrane domains that in CFTR open and close the ion pore. We have proposed that once ATP has bound to both NBDs of a closed CFTR channel the NBDs dimerize, burying the ATPs in the dimer interface, prompting channel opening: hydrolysis of one of the ATPs leads to disruption of the NBD dimer, and to channel closing. To further clarify this gating cycle, the goal of the research proposed here is to use single-channel kinetic analysis to identify all conformational states of a CFTR channel and the connectivities among them. We will use a novel statistical analysis we recently developed to dissect the two consecutive steps expected to occur in every open burst of a CFTR channel. To learn the molecular structural underpinnings of those steps, we will apply our analysis to wild-type and to select mutant CFTR channels while varying temperature, ionic strength, degree of regulatory PKA phosphorylation, and structure of activating nucleotide (i.e. ATP and/or analogs). We will also evaluate the newly uncovered complex gating of a non-hydrolytic mutant CFTR (D1370N). Finally, we will determine the molecular basis for the locked-open conformation of CFTR channels observed after exposing them to a mixture of ATP plus non-hydrolyzable ATP analog (e.g. AMPPNP). We will record from inside-out patches, excised from Xenopus oocytes expressing CFTR, in a fast-flow system with accurate temperature control.